preparing and evaluating blood smears

Question 1

Why make a proper blood smear

Answer 1

A differential WBC count can be done
You will be able to calculate the relative number of each type of WBC
RBC estimation and morphology can be evaluated
Platelet estimation and morphology can be evaluated
Identify blood parasites or other organisms

Question 2

What types of blood smears can be made

Answer 2

Wedge technique– we will use this one in the lab
Coverslip technique–mostly used for exotics
The smear should be made as soon as possible after collecting the sample

Question 3

Coverslip technique is

Answer 3

A drop of blood is placed on a coverslip
A second coverslip is placed diagonally on top of the first
Once the area between the coverslips is almost completely filled with blood, separate the coverslips in a smooth motion
Allow to air dry

Question 4

Wedge technique is

Answer 4

This is the most commonly used technique
Good quality smears are required to generate accurate results
Be sure your slides are clean and dry
Don’t touch the flat surfaces of the slides with your fingers
Use one drop of fresh whole blood if possible, if not use EDTA as the anticoagulant
Mix the sample well just before drawing up the sample
We will use a capillary tube to transfer a drop of blood to the slide
Label the slide with the patient’s name and date on the frosted edge
Select a second slide (spreader slide)– ensure that it does not have any chips or burrs on the short edge of the slide
Place a drop of blood near the frosted end of the slide
Hold the spreader slide at a 30° to 45 ° angle to the table
The higher the angle the shorter the smear
Place your index and middle fingers on the frosted end of the bottom slide to immobilize it
You may choose to use your thumb while holding the edge of the counter
Pull the short end of the spreader slide into the drop of blood
Once the blood has spread approximately 2/3 to 3/4 of the width of the spreader slide, push the spreader slide across the bottom slide in a swift, steady, straight motion
Do not lift the spreader slide until you have gone past the end of the bottom slide

Question 5

What should a proper blood smear look like

Answer 5

You want a flame/thumb shaped smear of good size that is neither too thin nor too thick
Want to have a large monolayer
Monolayer- one cell layer thick
Gently wave the smear in the air to dry it rapidly
This prevents artifacts from forming
If the smear isn’t the correct shape, it will alter cell distribution and therefore the cell counts

Question 6

Problems with blood smears

Answer 6

Artifacts will develop if the slide dries too slowly
If the smear is thin– the angle may have been <30°, it was spread too quickly, or the patient is anemic
Cells will be crushed and distorted
If smear is thick-angle >45°, too much blood was used, or the patient is hemoconcentrated
WBCs will appear small & dark, cells will be overlapping and difficult to evaluate, the monolayer will be thin
If you push to slow-the segmented WBCs & monocytes will be concentrated at the feathered edge and there will be distortion of the cells

Question 7

Staining on the blood smear

Answer 7

Staining helps to differentiate the different WBC types and aid in the detection of any abnormalities that may be present
Performed once the slide has completely dried
Any Romanowsky stain can be used
Includes Wright’s stain, Giemsa stain or Modified Wright’s stain
Available in a one- step or three-step process
Diff-Quik is the most common– this is what we will use in the lab
Make sure to wear gloves while staining slides
The last step in any staining procedure is to rinse with distilled water
The slide must be completely dry before it is evaluated with the microscope
Always evaluate the slide grossly before looking at it with the microscope, thai will allow you to determine if the slide was prepared properly
Look at the size and shape of the blood smear
Evaluate the monolayer
Look for evidence of air bubble, streaks or stain precipitate within the smear
Evaluate the intensity of the staining

Question 8

Staining with modified wright’s stains (manufacturers recommendation)

Answer 8

Fast, easy and consistent
Dip dry smear in solution 1
5- 1 second dips
Repeat for solutions 2 and 3
Then rinse the slide is distilled water
Let the slide dry with the feathered edge up
Microscopic examination of the slide when it’s completely dry

Question 9

Staining problems

Answer 9

Over staining
Over rinsing
Not letting the smear dry properly
Old, dirty stain –don’t add stain to jars, change it!
Water contamination of the fixative